1. Field of the Invention
The invention concerns the identification of proteomes of biological fluids and their use in determining the state of maternal/fetal conditions, including maternal conditions of fetal origin, chromosomal aneuploidies, and fetal diseases associated with fetal growth and maturation. In particular, the invention concerns a comprehensive proteomic analysis of human amniotic fluid (AF) and cervical vaginal fluid (CVF), and the correlation of characteristic changes in the normal proteome with various pathologic maternal/fetal conditions, such as intra-amniotic infection, pre-term labor, and/or chromosomal defects. The invention further concerns the identification of biomarkers and groups of biomarkers that can be used for non-invasive diagnosis of various pregnancy-related disorders, and diagnostic assays using such biomarkers.
2. Description of the Related Art
Proteomics
The large-scale analysis of protein expression patterns is emerging as an important and necessary complement to current DNA cloning and gene profiling approaches (Pandey and Mann, Nature 405:837-46 (2000)). DNA sequence information is helpful in deducing some structural and potential protein modifications based on homology methods, but it does not provide information on regulation of protein function through post-translational modifications, proteolysis or compartmentalization.
Traditional gel-based methods, such as one- and two-dimensional gel electrophoresis are useful for small-scale protein detection (<1,000 proteins), but these require large sample quantity (Lilley K S, Razzaq A, Dupree P: Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Curr Opin Chem Biol. 6(1):46-50, 2002). Approaches to overcome this limitation include matrix-assisted or surface-enhanced laser desorption/ionization (MALDI or SELDI) time-of-flight mass spectrometers that accurately generate profiles showing the masses of proteins in a sample. These patterns or profiles can be used to identify and monitor various diseases. The second level of identification comes from coupling peptide mapping to tandem mass spectrometry to generate amino acid sequence information from peptide fragments. This can, for example, be achieved by coupling the MALDI/SELDI or ESI to quadrupole time-of-flight. MS (Qq-TOF MS). The latter method can also be used for quantification of specific peptides (ICAT technology).
Diagnosis of Pathologic Maternal/Fetal Conditions
There are numerous pathologic maternal and fetal conditions, such as intra-amniotic infection (IAI), preeclampsia, preterm delivery and labor, and chromosomal aneuploidies, that may develop during pregnancy and compromise the well-being or, in some instances, threaten the life of the mother and/or the fetus or newborn. Early diagnosis of such conditions is critical to allow timely treatment and intervention. Unfortunately, early diagnosis for most of these conditions is difficult because the clinical signs and symptoms occur late, and are often non-specific and inconsistent. For example, the clinical symptoms of IAI typically include maternal fever and leukocytosis, but these symptoms often occur later and are neither sensitive nor specific. Thus, Gravett et al., Am. J. Obstet. Gynecol. 171:1660-7 (1994), utilizing a non-human primate model, demonstrated that following experimental intra-amniotic infection with Group B streptococcus, fever and leukocytosis are present only 50% of the time at the onset of infection-induced preterm labor, which occurs 28 to 40 hours after experimental infection. Therefore, to avoid a delay in diagnosis, a high index of suspicion and the appropriate use of adjunctive laboratory tests, are warranted. The clinical criteria commonly used to diagnose IAI include maternal fever. (≧37.8° C.), along with two or more of the following: maternal leukocytosis (≧15,000/mm3), maternal or fetal tachycardia, uterine tenderness, or foul-smelling amniotic fluid.
Because of the inconsistency of clinical features, other adjunctive laboratory tests have been utilized to aid in the diagnosis of IAI. These include: measurement of maternal C-reactive protein, direct examination of amniotic fluid for leukocytes or bacteria on Gram stain, amniotic fluid culture, measurement of amniotic fluid glucose concentrations, detection of amniotic fluid leukocyte esterase, detection of bacterial organic acids by gas-liquid chromatography, measurements of various amniotic fluid or vaginal cytokines (e.g., interleukins 2, 4, 6, granulocyte colony-stimulating factor, and tumor necrosis factor-α), matrix metalloproteinase-9, lactoferrin, and assessment of fetal activity (biophysical profile) by ultrasonography. Measurement of cytokines or other biochemical factors is expensive, generally not clinically available, and is primarily a research tool. Further, the testing efficiency of these tests has not been consistently better than more readily available traditional tests such as amniotic fluid Gram stain and culture, amniotic fluid glucose concentrations, and detection of amniotic fluid leukocyte esterase. The efficiency of these tests has been previously extensively reviewed. (Ohlsson, A. and Wang, E.: An analysis of antenatal tests to detect infection at preterm rupture of the membranes. American Journal of Obstetrics and Gynecology 162:809, 1990). Although all have reasonable sensitivity, specificity, and predictive value none are sufficiently sensitive or specific to be utilized independently of clinical features in the diagnosis of IAI.
Accordingly, there is a great need for new approaches that allow early and accurate diagnosis of IAI and other pathologic maternal/fetal conditions, especially pre-term labor and delivery.
It is particularly desirable to develop new, efficient and reliable non-invasive methods for the diagnosis of chromosomal aneuploidies. At present the definitive diagnosis of chromosomal aneuploidies following maternal serum screening and ultrasound requires a mid-trimester genetic amniocentesis. This is an invasive procedure associated with a 0.5% risk of loss of the pregnancy. Further, chromosomal analysis of amniotic fluid cells is a labor-intensive and time-consuming procedure, taking up to 2 weeks. Reliable tests are therefore necessary to improve the detection of chromosomal aneuploidies from maternal serum, or other biological fluids, reduce the unacceptably high false positive rate of maternal screening, and increase the speed and efficiency of diagnosis from amniotic fluid following amniocentesis. Other pathologic aneuploidic conditions, such as Klinefelter syndrome and Turner syndrome, may be entirely missed by screening with ultrasonography or conventional maternal serum screening.